1. Field of the Invention
The ability to specifically cleave a duplex DNA molecule into discrete fragments is essential for the manipulation and modification of DNA in vitro. Such specific cleavage is accomplished using restriction endonucleases which are enzymes capable of cutting a DNA molecule at or about a specific recognition site. Type II restriction endonucleases recognize and cleave DNA at particular sequences of from four to seven nucleotides which usually have an axis of rotational symmetry. The site of cleavage may be within the recognition sequence or may lie a fixed number of base pairs away from the sequence. Moreover, the cleavage may be cut straight across the duplex producing flush or blunt-ended fragments, or may be staggered to produce either 5'- or 3'- cohesive ends.
Isoschizomers are different restriction enzymes which recognize the same target sequence and which may share a common cleavage pattern. Often, however, the isoschizomers will be affected differently by methylation of the substrate DNA. For example, while one member of an isoschizomer pair may be inhibited by methylation of a particular cytosine residue in the recognition sequence, the other member of the pair may be unaffected. Thus, two isoschizomers may produce different restriction patterns, depending on methylation of the DNA being cleaved.
To perform genetic manipulation in vitro, it is desirable to have a very large number of restriction endonucleases available to perform cleavage at preselected locations. Under certain circumstances, the use of one isoschizomer may be favored over another because of different cleavage patterns brought about by methylation of the substrate DNA. Moreover, one isoschizomer may be preferred over another because of greater inherent stability, greater purity from other contaminating enzymes, and the like.
2. Description of the Prior Art
Many restriction enzymes are reported in the literature, and a number of the reported enzymes are commercially available. Restriction enzyme XmaI recognizes the sequence 5'-CCCGGG-3' and cleaves between the first and second residues to produce a 5'-CCGG cohesive end. The isolation and use of XmaI are described by Endow and Roberts (1977) J. Mol. Biol. 112:521-529 and Kunkel et al. (1979) J. Mol. Biol. 132:133-139. The purification of XmaI is complicated by the presence of two other restriction enzymes, XmaII and XmaIII, and preparations of XmaI are often contaminated by these other enzyme activities. Moreover, XmaI is insufficiently stable to allow long-term storage and routine use.